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The Blood Sugar Response of Turkey Poults to Insulin*
The Blood Sugar Response of Turkey Poults to Insulin* JAMES G. SNEDECOR, HARRIET MATHEWS AND WALTER B. MACGRATH, J R .
Department of Zoology, University of Massachusetts, Amherst (Received for publication August 18, 1955)
/ " \ UR interest in the blood sugar response Indications of hyperglycemic responses, ^ ^ of poults to insulin (Snedecor, 1952) on the other hand, were noted by Honeyarose from studies on insulin resistance in well and Riddle (1923) who obtained (folmice (Chase et al., 1948; MacGrath and lowing initial hypoglycemia) both hypoSnedecor, 1953). Birds in general fre- and hyperglycemic effects in different quently have been reported to be much pigeons 21 hours after single doses of insulin more resistant to insulin than are mammals. (ranging from 10 to 30 units/kg.). Riddle This resistance, however, must be qualified and Opdyke (1942) and Opdyke (1942) also depending upon dosage and criteria of observed hyperglycemic responses. Opdyke response. (1942) found that, in 30-40 day old chicks Chen, Anderson and Maze (1945) showed treated daily with 60 or 120 units per kilothat the median convulsive doses of insulin gram, the blood sugars taken daily before for the duck, pigeon, and canary were 157, the next injections showed a gradual but 705, and 2,396 units per kilogram as com- decided rise, and liver glycogen also rose pared with 5 for the rabbit and about 20 above the control level. Chen et al. (1945) for mouse, rat, and dog. Roosters showed no reported blood sugar curves for four convulsions even at 5,000 units per kilo- roosters that received 200-5,000 units per gram. Convulsions seem a less reliable kilogram, and in no case was there a criterion of insulin effect in birds than in marked hypoglycemia during the first seven mammals as birds frequently may succumb hours following injection. In general, the to insulin without showing any convulsive higher the insulin dose the higher the blood sugar remained. behavior. When blood sugar responses have been The present study was concerned with employed as a criterion for insulin effect, the blood sugar responses of day old poults evidence for resistance depends upon the to a wide range of insulin dosages. dosage employed. Thus, Corkill (1930) METHODS observed a definite hypoglycemia in fasted The Jersey Buff poults used in these chickens (3 months of age) given 80 units per bird in hourly doses of 20 units. Golden studies were obtained from the Poultry and Long (1942), and Opdyke (1942) also Department of the University of Massareported a definite hypoglycemia for chicks chusetts. A total of 335 poults was used. following insulin doses in the neighborhood They were placed in chick boxes upon of one to five units per kilogram with a hatching and kept without food or water return to normal in four to five hours, but until they were used on the following day. with no convulsions. Golden and Long Insulin injections were made subcutanoted that there was very little difference neoulsy and the birds were returned to the in effect between their small and Corkill's boxes after injection. Blood was obtained by decapitating the much larger insulin dosage. birds and collecting several drops of blood * Investigation aided and insulin generously on a spot plate. No anticoagulant was used. A 0.1 ml. sample was drawn from the desupplied by Eli Lilly and Company. 355
356
J. G. SNEDECOR, H. MATHEWS AND W. B. MACGRATH, JR.
pression and added to 1.9 cc. of the deproteinizing reagent (0.5% Na tungstate in N/30 H2S04) in a centrifuge tube. The tube was allowed to stand for five minutes, then centrifuged. One ml. of the clear supernatent was analyzed for sugar according to the method of Nelson (1944). The poults were of fairly uniform weight, and for convenience all birds in a group received the same unitage of insulin rather than according to weight. The birds were assigned to groups of 5 or 6 in random fashion. The sex of the birds was not determined. All insulin dosages from 25 to 100 units were made directly with an insulin solution containing 500 units per ml. Doses of 10 units or less were made from preparations containing 100 or 40 units/cc. diluted where necessary with 0.2% phenol at a pH of 3.5 The glucagon (hyperglycemicglycogenolytic material) was prepared by incubating regular U-500 insulin with alkali according to the method of Sutherland and Cori (1948). RESULTS
In the initial series of experiments insulin dosages of one, and 100 units per bird (approximately 20 and 2,000 u./kg.) were used. Groups were run for each dosage at hourly intervals for five hours following insulin. A prolonged hyperglycemia persisted for the five hours in the hundred unit series, the maximum being at 4 hours after the injection. The curve based on groups receiving one unit showed a definite hypoglycemia throughout the five hours (Fig. 1). The small number of birds available from the second hatch limited the trials to one series at three units and a single group at 100 units. Again the small dose produced hypoglycemia from the first hour while the single 100 unit value at two hours showed hyperglycemia comparable to that produced in the first 100 unit series. The data
HATCH I 8 2 HOURS AFTER
INJECTION
FIG. 1. Blood sugar curves resulting from the subcutaneous injection of insulin in day-old turkeys. Each point on the graph represents an average of five determinations. The stippled areas indicate ± standard error. In the case of the one unit and the 3 unit curves, since the birds were from different hatches, the standard errors are only to indicate the variability within groups, and no implication of statistical differences is intended.
for all series are summarized in Table 1. In hatch 3 birds, insulin dosages below 25 units (approximately 500 units per kg.) did not produce the hyperglycemic response seen at higher dosages, and for the first hour an inverse relationship between dosage and blood sugar depression existed. The lower the insulin dose, the more profound was the hypoglycemia. The same relationship held also for the second hour with the exception of the 0.1 unit (2 units per kg.) value which was above the six unit value but not above the ten unit value. It should be noted that the 25 and 50 unit series of hatch 3 were run on two-day-old birds. Whether or not the two-day-old bird
Insulin Glucagon
Glucagon-free insulin
Insulin
Injection 2,000 20 2,000 60 200 120 2 1,000 500 2,000 1,000 200 2,000
Approx. units/kg.
100 2,000 100 2,000 100 2,000 (Equivalent)
100 1 100 3 10 6 0.1 50* 25* 100 50 10 100
Units per poult
* Two day old birds. t Standard error. % Number dead in group (no blood sugar determination).
5 (July)
4 (July)
3 (June)
2 (May)
1 (April)
Hatch No. and month
—
107 + 4.7f 54±2.1
1 hour
100 + 4.5
60 + 6.9 137 + 7.7 117 + 4.8
78 + 2.7 100 + 8.7 87 + 6.3 81 + 3.3 67 + 4.9 100 + 5.9 * 142 + 9.0 118 + 4.8 100 + 3.5 92 + 5.0 71+9.8 74+4.8 57±3.3
100 + 6,4
100 + 4.3
Control
60+ 3.9 137 + 18.7 126+ 6.5
131± 7.4 49+ 2.7 125± 1.8 61+ 2.8 67 ± 1 9 . 0 44± 4.3 63± 7.4 116+14.6 79± 9.2 93 + 10.0 46± 7.6 52+ 6.5 50± 4.2
2 hours
•
4.0 3.7(-l)J 2.0 3.9 5.0 4.7 9.8 4.1 2.2 6.4 52+ 5.8 74+19.7 149 + 11.5
55± 74± 56+ 70+ 88± 54+ 98+ 55± 57± 40+
—
123 + 13.6 3 4 ± 4.2
3 hours
% of control blood sugar
132 + 5.7
—. —.
37 + 7.1 34±2.9 52 + 1 . 6 ( - l ) 51±4.5
— —
—.
54+3.5 46 + 5.5 73±4.4 46±6.4
143 + 8.1 32+4.3
4 hours
5 hours
22 + 2.1 44±4.4 97 + 7.6
30 + 2.9 30 + 2.8 27 + 3.3 ( - 2 ) 56+3.7
—
—
35±2.1 42 + 5.0 53 + 6.0 62+4.9 60 + 4.7 ( - 1 )
121 + 9.8 21+4.7
TABLE 1.—Blood sugar response of day-old poults to various doses of insulin. Averages are of groups of five birds (6 in hatches 4 and 5)
Co Cn —J
§
Z d tr1
h-1
H O
en
H
d
o
H-l
H O **1
t/5
H TJ O
358
J. G. SNEDECOR, H. MATHEWS AND W. B. MACGRATH, J R .
responds differently from the one-day-old we do not know. The 100 unit series of the fourth hatch showed no hyperglycemia; however, the blood sugar level was only slightly below the control level for three hours, dropping off rapidly then to definitely hypoglycemic values. The absence of a hyperglycemic phase in this series possibly is due to a response difference on the part of this particular hatch of poults. In all the series run on birds from hatch 4 the blood sugar values were definitely lower than for comparable doses on other hatches of birds. A third 100 unit series was run later (hatch 5) with results more like the initial 100 unit series except that the hyperglycemic phase was only between two and three hours in duration (Fig. 2).
It seemed possible that the prolonged hyperglycemic phase might be due to glucagon (which accompanies the regular insulin). Therefore, two 100 unit series were run (hatches 4 and 5) using insulin free of glucagon (Lilly, 499667). Figure 2 illustrates that in such series no hyperglycemic phase was present, strongly suggesting that the glucagon indeed was responsible for the variable hyperglycemic effect of large insulin doses. To determine the effect of glucagon alone, an amount approximately equivalent to that in 100 units of regular insulin was used and the resulting curve (shown in Fig. 2) indicated that the blood sugar level remained well above the control level for at least four hours and further substantiated the idea that this amount of glucagon could be responsible for a hyperglycemia of several hours duration. DISCUSSION
HATCH
5
HOURS
AFTER
INJECTION
FIG. 2. Comparison of the effects of subcutaneous injection of regular insulin, glucagon-free insulin and glucagon upon the blood sugar level of day-old poults. Stippled areas represent ± standard error.
The prolonged hyperglycemic effect of large doses of insulin in the poult was very striking, and evidently was due to the presence of glucagon in the insulin preparation, since insulin free of glucagon had no hyperglycemic effect. Previous work with mice (MacGrath and Snedecor, 1953) showed that more glucagon was extractable from the pancreas following large doses of insulin than normally. Vuylsteke and de Duve (1953) have reported that the bird pancreas contains six times as much glucagon as the mammalian pancreas. We have wondered if endogenously produced glucagon could have played a role in the poult reponse to insulin. It is of interest to note, in this connection, that Jourdonais and Burger (1940) reported hyperglycemia in four out of nine cases in which rabbits were given 1,000 unit doses of insulin, and, further, they were able to extract a hyperglycemic material from the blood of the rabbits as
RESPONSE OF POULTS TO INSULIN
long as 90-180 minutes after the insulin injection when insulin was no longer recoverable. The extraction was designed to recover insulin and it is possible that glucagon was the hyperglycemic substance they obtained. In fact, Jourdonais and Bruger suggested it might be an endogenously produced anti-insulin substance. It seems unlikely that exogenous glucagon would have been present so long after injection, for, as Sutherland and de Duve (1948) indicated, glucagon is rapidly destroyed in the circulating blood (mammal). The massive dose of 1,000 units used by Jourdonais and Bruger, approximates the doses (500-2,000 u./kg.) which produced hyperglycemia in the present study. Whether or not the glucagon is entirely of exogenous origin does not alter the surprising fact of the long hyperglycemic period produced by injected glucagon and by insulin containing glucagon. The question arises as to how glucagon can produce such a long lasting hyperglycemia. It seems unlikely that liver glycogen could maintain such a prolonged rise in blood sugar. Perhaps glucagon has a function in the poult not present or not yet detected in the mammal, e.g., some effect upon gluconeogenesis. In mammals, the hyperglycemia following glucagon injection rarely lasts longer than 30-40 minutes, and in the presence of insulin the hyperglycemic phase is invariably shorter than with glucagon alone —rarely longer than 20 minutes.Furthermore, the hyperglycemic response to glucagon is usually not apparent following subcutaneous injection because the glucagon evidently is destroyed rapidly by mammalian tissues. The persistence of the glucagon effect in the poult following subcutaneous injection indicates that glucagon is not rapidly destroyed before entering the circulation. This may, in
359
part, explain the duration of the hyperglycemic effect. There may be factors in addition to glucagon which are responsible for high blood sugar following insulin. Riddle, Honeywell and Fisher (1924) noted adrenal enlargement resulting from large doses of insulin in pigeons and doves. They were inclined toward the idea that this represented an increased epinephrine output, but Miller and Riddle (1941) showed that a decided response of the cortical cells occurred in response to insulin. The changes were comparable to those elicited by adrenocorticotrophin administration. Golden and Long (1942) showed that chicks, like mammals, respond to adrenal cortical extracts by a rise in blood sugar and increased liver glycogen. The extent to which cortical secretions play a role in the response of poults to insulin has not been investigated. SUMMARY
The effect of various doses of insulin upon the blood sugar of day-old turkey poults has been investigated. The blood sugar changes were followed for five hours. Below about 25 units per bird (approximately 500 units per kilogram) the curve dropped below the normal level and did not again reach the control level during the five hours. At a dose of 25 units per bird or higher, the blood sugar, in most cases, rose and remained above the control level for varying lengths of time. At 100 units per bird, the curves have indicated hyperglycemia of two to five hours duration. Comparison of the effects of regular insulin with glucagon-f ree insulin and with an amount of glucagon equal to that in the regular insulin showed that the hyperglycemia was satisfactorily explained by the presence of glucagon in the regular insulin preparation. The prolonged effect
360
J . R . SlZEMORE AND R . J . LlLLIE
of glucagon in the poult indicated an action somewhat different from that of glucagon in mammals. REFERENCES Chase, H . B., M. S. Gunther, J. Miller and D. Wolffson, 1948. High insulin tolerance in an inbred strain of mice. Science, 107: 408-411. Chen, K. K., R. C. Anderson and N . Maze, 1945. Susceptibility of birds to insulin as compared with mammals. J. Pharm. Exper. Therap. 84: 74-76. Corkill, B., 1930. The influence of insulin on the distribution of glycogen in the normal animal. Biochem. J. 24: 779. Golden, W. R. C , and C. N. H. Long, 1942. The influence of certain hormones on the carbohydrate levels of the chick. Endocrinol. 3 : 675-686. Honeywell, H . E., and O. Riddle, 1923. The action of Iletin (insulin) on the blood sugar of pigeons. Proc. Soc. Exp. Biol. Med. 20: 248-252. Jourdonais, L. F., and M. Bruger, 1940. The sojourn of insulin in the blood of rabbits after the administration of massive doses of insulin. Endocrinol. 26: 250-254. MacGrath, W. B., Jr., and J. G. Snedecor, 1953. The effect of insulin on the glucagon content of the
pancreas. Diabetes, 2:443-447. Miller, R. A., and O. Riddle, 1941. Cellular response to insulin in suprarenals of pigeons. Proc. Soc. Exp. Biol. Med. 47:449-453. Nelson, N., 1944. A photometric adaptation of the Somogyi method for the determination of glucose. J. Biol. Chem. 153: 375-380. Opdyke, D. F., 1942. Response of fasted and nonfasted chicks to insulin. Endocrinol. 31:363-370. Riddle, O., H. E. Honeywell and W. S. Fisher, 1924. Suprarenal enlargement under heavy dosage with insulin. Am. J. Physiol. 68:461-476. Riddle, O., and D . F. Opdyke, 1942. Gross differences in response of liver to first and later daily dosage with insulin and prolactin. Fed. Proc. 1: 72. Snedecor, J. G., 1952. The blood sugar response of poults to insulin. Anat. Rec. 113: 566. Sutherland, E. W., and C. F. Cori, 1948. Influence of insulin preparations on glycogenolysis in liver slices. J. Biol. Chem. 172: 737-750. Sutherland, E. W-, and C. de Duve, 1948. Origin and distribution of the hyperglycemic-glycogenolytic factor of the pancreas. J. Biol. Chem. 663-674. Vuylsteke, C. A., and C. de Duve, 1953. Le contenu en glucagon du pancreas avaiare. Arch, iriternat. physiol. 61:273-275.
Lack of Effect of a Synthetic Poultry Feed Flavor on Chick Growth and Feed Efficiency J . R . SlZEMORE AND R . J . LlLLIE Poultry Section, Animal and Poultry Husbandry Research Branch, Agricultural Research Service, U. S. Department of Agriculture, Beltsville, Maryland (Received for publication August 18, 1955)
A
SERIES of three feeding trials of four •weeks duration were run to determine whether a synthetic poultry feed flavor had any influence upon chick growth and feed efficiency when incorporated into a practical chick starting ration. The poultry feed flavor contained extractives of anise, rose, cinnamon, lemon and orris with soya meal and corn sugar used as carriers. The experimental design is presented in Table 1. Fish meal was added at the 4 percent level at the expense of the total ration
whereas 4 percent of pork fat was added at the expense of corn and the protein adjusted by increasing the soybean meal. The synthetic flavor was added at the rates of f, J, and 1 pound per ton (125 ppm., 250 ppm., and 500 ppm. respectively). Day-old straight run New Hampshire X Silver Cornish chicks were distributed into groups of 30 chicks each in experiments 1 and 2, and in groups of 15 each in experiment 3. The chicks were wing banded and weighed individually at hatching time.
Department of Zoology, University of Massachusetts, Amherst (Received for publication August 18, 1955)
/ " \ UR interest in the blood sugar response Indications of hyperglycemic responses, ^ ^ of poults to insulin (Snedecor, 1952) on the other hand, were noted by Honeyarose from studies on insulin resistance in well and Riddle (1923) who obtained (folmice (Chase et al., 1948; MacGrath and lowing initial hypoglycemia) both hypoSnedecor, 1953). Birds in general fre- and hyperglycemic effects in different quently have been reported to be much pigeons 21 hours after single doses of insulin more resistant to insulin than are mammals. (ranging from 10 to 30 units/kg.). Riddle This resistance, however, must be qualified and Opdyke (1942) and Opdyke (1942) also depending upon dosage and criteria of observed hyperglycemic responses. Opdyke response. (1942) found that, in 30-40 day old chicks Chen, Anderson and Maze (1945) showed treated daily with 60 or 120 units per kilothat the median convulsive doses of insulin gram, the blood sugars taken daily before for the duck, pigeon, and canary were 157, the next injections showed a gradual but 705, and 2,396 units per kilogram as com- decided rise, and liver glycogen also rose pared with 5 for the rabbit and about 20 above the control level. Chen et al. (1945) for mouse, rat, and dog. Roosters showed no reported blood sugar curves for four convulsions even at 5,000 units per kilo- roosters that received 200-5,000 units per gram. Convulsions seem a less reliable kilogram, and in no case was there a criterion of insulin effect in birds than in marked hypoglycemia during the first seven mammals as birds frequently may succumb hours following injection. In general, the to insulin without showing any convulsive higher the insulin dose the higher the blood sugar remained. behavior. When blood sugar responses have been The present study was concerned with employed as a criterion for insulin effect, the blood sugar responses of day old poults evidence for resistance depends upon the to a wide range of insulin dosages. dosage employed. Thus, Corkill (1930) METHODS observed a definite hypoglycemia in fasted The Jersey Buff poults used in these chickens (3 months of age) given 80 units per bird in hourly doses of 20 units. Golden studies were obtained from the Poultry and Long (1942), and Opdyke (1942) also Department of the University of Massareported a definite hypoglycemia for chicks chusetts. A total of 335 poults was used. following insulin doses in the neighborhood They were placed in chick boxes upon of one to five units per kilogram with a hatching and kept without food or water return to normal in four to five hours, but until they were used on the following day. with no convulsions. Golden and Long Insulin injections were made subcutanoted that there was very little difference neoulsy and the birds were returned to the in effect between their small and Corkill's boxes after injection. Blood was obtained by decapitating the much larger insulin dosage. birds and collecting several drops of blood * Investigation aided and insulin generously on a spot plate. No anticoagulant was used. A 0.1 ml. sample was drawn from the desupplied by Eli Lilly and Company. 355
356
J. G. SNEDECOR, H. MATHEWS AND W. B. MACGRATH, JR.
pression and added to 1.9 cc. of the deproteinizing reagent (0.5% Na tungstate in N/30 H2S04) in a centrifuge tube. The tube was allowed to stand for five minutes, then centrifuged. One ml. of the clear supernatent was analyzed for sugar according to the method of Nelson (1944). The poults were of fairly uniform weight, and for convenience all birds in a group received the same unitage of insulin rather than according to weight. The birds were assigned to groups of 5 or 6 in random fashion. The sex of the birds was not determined. All insulin dosages from 25 to 100 units were made directly with an insulin solution containing 500 units per ml. Doses of 10 units or less were made from preparations containing 100 or 40 units/cc. diluted where necessary with 0.2% phenol at a pH of 3.5 The glucagon (hyperglycemicglycogenolytic material) was prepared by incubating regular U-500 insulin with alkali according to the method of Sutherland and Cori (1948). RESULTS
In the initial series of experiments insulin dosages of one, and 100 units per bird (approximately 20 and 2,000 u./kg.) were used. Groups were run for each dosage at hourly intervals for five hours following insulin. A prolonged hyperglycemia persisted for the five hours in the hundred unit series, the maximum being at 4 hours after the injection. The curve based on groups receiving one unit showed a definite hypoglycemia throughout the five hours (Fig. 1). The small number of birds available from the second hatch limited the trials to one series at three units and a single group at 100 units. Again the small dose produced hypoglycemia from the first hour while the single 100 unit value at two hours showed hyperglycemia comparable to that produced in the first 100 unit series. The data
HATCH I 8 2 HOURS AFTER
INJECTION
FIG. 1. Blood sugar curves resulting from the subcutaneous injection of insulin in day-old turkeys. Each point on the graph represents an average of five determinations. The stippled areas indicate ± standard error. In the case of the one unit and the 3 unit curves, since the birds were from different hatches, the standard errors are only to indicate the variability within groups, and no implication of statistical differences is intended.
for all series are summarized in Table 1. In hatch 3 birds, insulin dosages below 25 units (approximately 500 units per kg.) did not produce the hyperglycemic response seen at higher dosages, and for the first hour an inverse relationship between dosage and blood sugar depression existed. The lower the insulin dose, the more profound was the hypoglycemia. The same relationship held also for the second hour with the exception of the 0.1 unit (2 units per kg.) value which was above the six unit value but not above the ten unit value. It should be noted that the 25 and 50 unit series of hatch 3 were run on two-day-old birds. Whether or not the two-day-old bird
Insulin Glucagon
Glucagon-free insulin
Insulin
Injection 2,000 20 2,000 60 200 120 2 1,000 500 2,000 1,000 200 2,000
Approx. units/kg.
100 2,000 100 2,000 100 2,000 (Equivalent)
100 1 100 3 10 6 0.1 50* 25* 100 50 10 100
Units per poult
* Two day old birds. t Standard error. % Number dead in group (no blood sugar determination).
5 (July)
4 (July)
3 (June)
2 (May)
1 (April)
Hatch No. and month
—
107 + 4.7f 54±2.1
1 hour
100 + 4.5
60 + 6.9 137 + 7.7 117 + 4.8
78 + 2.7 100 + 8.7 87 + 6.3 81 + 3.3 67 + 4.9 100 + 5.9 * 142 + 9.0 118 + 4.8 100 + 3.5 92 + 5.0 71+9.8 74+4.8 57±3.3
100 + 6,4
100 + 4.3
Control
60+ 3.9 137 + 18.7 126+ 6.5
131± 7.4 49+ 2.7 125± 1.8 61+ 2.8 67 ± 1 9 . 0 44± 4.3 63± 7.4 116+14.6 79± 9.2 93 + 10.0 46± 7.6 52+ 6.5 50± 4.2
2 hours
•
4.0 3.7(-l)J 2.0 3.9 5.0 4.7 9.8 4.1 2.2 6.4 52+ 5.8 74+19.7 149 + 11.5
55± 74± 56+ 70+ 88± 54+ 98+ 55± 57± 40+
—
123 + 13.6 3 4 ± 4.2
3 hours
% of control blood sugar
132 + 5.7
—. —.
37 + 7.1 34±2.9 52 + 1 . 6 ( - l ) 51±4.5
— —
—.
54+3.5 46 + 5.5 73±4.4 46±6.4
143 + 8.1 32+4.3
4 hours
5 hours
22 + 2.1 44±4.4 97 + 7.6
30 + 2.9 30 + 2.8 27 + 3.3 ( - 2 ) 56+3.7
—
—
35±2.1 42 + 5.0 53 + 6.0 62+4.9 60 + 4.7 ( - 1 )
121 + 9.8 21+4.7
TABLE 1.—Blood sugar response of day-old poults to various doses of insulin. Averages are of groups of five birds (6 in hatches 4 and 5)
Co Cn —J
§
Z d tr1
h-1
H O
en
H
d
o
H-l
H O **1
t/5
H TJ O
358
J. G. SNEDECOR, H. MATHEWS AND W. B. MACGRATH, J R .
responds differently from the one-day-old we do not know. The 100 unit series of the fourth hatch showed no hyperglycemia; however, the blood sugar level was only slightly below the control level for three hours, dropping off rapidly then to definitely hypoglycemic values. The absence of a hyperglycemic phase in this series possibly is due to a response difference on the part of this particular hatch of poults. In all the series run on birds from hatch 4 the blood sugar values were definitely lower than for comparable doses on other hatches of birds. A third 100 unit series was run later (hatch 5) with results more like the initial 100 unit series except that the hyperglycemic phase was only between two and three hours in duration (Fig. 2).
It seemed possible that the prolonged hyperglycemic phase might be due to glucagon (which accompanies the regular insulin). Therefore, two 100 unit series were run (hatches 4 and 5) using insulin free of glucagon (Lilly, 499667). Figure 2 illustrates that in such series no hyperglycemic phase was present, strongly suggesting that the glucagon indeed was responsible for the variable hyperglycemic effect of large insulin doses. To determine the effect of glucagon alone, an amount approximately equivalent to that in 100 units of regular insulin was used and the resulting curve (shown in Fig. 2) indicated that the blood sugar level remained well above the control level for at least four hours and further substantiated the idea that this amount of glucagon could be responsible for a hyperglycemia of several hours duration. DISCUSSION
HATCH
5
HOURS
AFTER
INJECTION
FIG. 2. Comparison of the effects of subcutaneous injection of regular insulin, glucagon-free insulin and glucagon upon the blood sugar level of day-old poults. Stippled areas represent ± standard error.
The prolonged hyperglycemic effect of large doses of insulin in the poult was very striking, and evidently was due to the presence of glucagon in the insulin preparation, since insulin free of glucagon had no hyperglycemic effect. Previous work with mice (MacGrath and Snedecor, 1953) showed that more glucagon was extractable from the pancreas following large doses of insulin than normally. Vuylsteke and de Duve (1953) have reported that the bird pancreas contains six times as much glucagon as the mammalian pancreas. We have wondered if endogenously produced glucagon could have played a role in the poult reponse to insulin. It is of interest to note, in this connection, that Jourdonais and Burger (1940) reported hyperglycemia in four out of nine cases in which rabbits were given 1,000 unit doses of insulin, and, further, they were able to extract a hyperglycemic material from the blood of the rabbits as
RESPONSE OF POULTS TO INSULIN
long as 90-180 minutes after the insulin injection when insulin was no longer recoverable. The extraction was designed to recover insulin and it is possible that glucagon was the hyperglycemic substance they obtained. In fact, Jourdonais and Bruger suggested it might be an endogenously produced anti-insulin substance. It seems unlikely that exogenous glucagon would have been present so long after injection, for, as Sutherland and de Duve (1948) indicated, glucagon is rapidly destroyed in the circulating blood (mammal). The massive dose of 1,000 units used by Jourdonais and Bruger, approximates the doses (500-2,000 u./kg.) which produced hyperglycemia in the present study. Whether or not the glucagon is entirely of exogenous origin does not alter the surprising fact of the long hyperglycemic period produced by injected glucagon and by insulin containing glucagon. The question arises as to how glucagon can produce such a long lasting hyperglycemia. It seems unlikely that liver glycogen could maintain such a prolonged rise in blood sugar. Perhaps glucagon has a function in the poult not present or not yet detected in the mammal, e.g., some effect upon gluconeogenesis. In mammals, the hyperglycemia following glucagon injection rarely lasts longer than 30-40 minutes, and in the presence of insulin the hyperglycemic phase is invariably shorter than with glucagon alone —rarely longer than 20 minutes.Furthermore, the hyperglycemic response to glucagon is usually not apparent following subcutaneous injection because the glucagon evidently is destroyed rapidly by mammalian tissues. The persistence of the glucagon effect in the poult following subcutaneous injection indicates that glucagon is not rapidly destroyed before entering the circulation. This may, in
359
part, explain the duration of the hyperglycemic effect. There may be factors in addition to glucagon which are responsible for high blood sugar following insulin. Riddle, Honeywell and Fisher (1924) noted adrenal enlargement resulting from large doses of insulin in pigeons and doves. They were inclined toward the idea that this represented an increased epinephrine output, but Miller and Riddle (1941) showed that a decided response of the cortical cells occurred in response to insulin. The changes were comparable to those elicited by adrenocorticotrophin administration. Golden and Long (1942) showed that chicks, like mammals, respond to adrenal cortical extracts by a rise in blood sugar and increased liver glycogen. The extent to which cortical secretions play a role in the response of poults to insulin has not been investigated. SUMMARY
The effect of various doses of insulin upon the blood sugar of day-old turkey poults has been investigated. The blood sugar changes were followed for five hours. Below about 25 units per bird (approximately 500 units per kilogram) the curve dropped below the normal level and did not again reach the control level during the five hours. At a dose of 25 units per bird or higher, the blood sugar, in most cases, rose and remained above the control level for varying lengths of time. At 100 units per bird, the curves have indicated hyperglycemia of two to five hours duration. Comparison of the effects of regular insulin with glucagon-f ree insulin and with an amount of glucagon equal to that in the regular insulin showed that the hyperglycemia was satisfactorily explained by the presence of glucagon in the regular insulin preparation. The prolonged effect
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of glucagon in the poult indicated an action somewhat different from that of glucagon in mammals. REFERENCES Chase, H . B., M. S. Gunther, J. Miller and D. Wolffson, 1948. High insulin tolerance in an inbred strain of mice. Science, 107: 408-411. Chen, K. K., R. C. Anderson and N . Maze, 1945. Susceptibility of birds to insulin as compared with mammals. J. Pharm. Exper. Therap. 84: 74-76. Corkill, B., 1930. The influence of insulin on the distribution of glycogen in the normal animal. Biochem. J. 24: 779. Golden, W. R. C , and C. N. H. Long, 1942. The influence of certain hormones on the carbohydrate levels of the chick. Endocrinol. 3 : 675-686. Honeywell, H . E., and O. Riddle, 1923. The action of Iletin (insulin) on the blood sugar of pigeons. Proc. Soc. Exp. Biol. Med. 20: 248-252. Jourdonais, L. F., and M. Bruger, 1940. The sojourn of insulin in the blood of rabbits after the administration of massive doses of insulin. Endocrinol. 26: 250-254. MacGrath, W. B., Jr., and J. G. Snedecor, 1953. The effect of insulin on the glucagon content of the
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Lack of Effect of a Synthetic Poultry Feed Flavor on Chick Growth and Feed Efficiency J . R . SlZEMORE AND R . J . LlLLIE Poultry Section, Animal and Poultry Husbandry Research Branch, Agricultural Research Service, U. S. Department of Agriculture, Beltsville, Maryland (Received for publication August 18, 1955)
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SERIES of three feeding trials of four •weeks duration were run to determine whether a synthetic poultry feed flavor had any influence upon chick growth and feed efficiency when incorporated into a practical chick starting ration. The poultry feed flavor contained extractives of anise, rose, cinnamon, lemon and orris with soya meal and corn sugar used as carriers. The experimental design is presented in Table 1. Fish meal was added at the 4 percent level at the expense of the total ration
whereas 4 percent of pork fat was added at the expense of corn and the protein adjusted by increasing the soybean meal. The synthetic flavor was added at the rates of f, J, and 1 pound per ton (125 ppm., 250 ppm., and 500 ppm. respectively). Day-old straight run New Hampshire X Silver Cornish chicks were distributed into groups of 30 chicks each in experiments 1 and 2, and in groups of 15 each in experiment 3. The chicks were wing banded and weighed individually at hatching time.